Homology modeling of Neurospora crassa geranylgeranyl pyrophosphate synthase: structural interpretation of mutant phenotypes.
A model of the tertiary structure of the Neurospora crassa carotenogenic prenyltransferase, geranylgeranyl pyrophosphate synthase (GGPPS), is presented, based on structural homology with other prenyltransferases and on the crystal structure of recombinant avian farnesyl pyrophosphate synthase (FPPS). The conserved aspartate-rich motifs DDxx(xx)D and associated basic residues, considered to be the active sites for binding and catalysis in all prenyltransferases, are highly conserved in the N. crassa GGPPS protein, while other regions display a lower degree of sequence homology; thus the GGPPS model structure is predicted to be highly reliable in the active site region. A number of carotene-deficient mutants have been generated utilizing the repeat-induced point mutation (RIP) mechanism: mutant al-3RIP1 carries a Ser-to-Asn mutation in position 336 which falls within the predicted active site of the enzyme. Analysis of the model structure of this mutant indicates that Ser336 may be involved in substrate uptake. Two other mutants, al-3RIP3 and al-3RIP6, carry mutations in positions in the GGPPS protein, homologous to regions of the avian FPPS enzyme proposed to be involved in enzyme dimerization and substrate uptake, respectively, suggesting an explanation for the reduced carotene content of these mutants.
Quondam
M
M
Dipartimento di Biopatologia Umana, Policlinico Umberto I, Università di Roma La Sapienza, Rome, Italy.
Barbato
C
C
Pickford
A
A
Helmer-Citterich
M
M
Macino
G
G
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010641:Phenotype; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012984:Software
D011994:Recombinant Proteins; D010088:Oxidoreductases; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase